DNA sequences encoding osteoinductive products

ABSTRACT

Purified BMP-2 proteins and processes for producing them are disclosed. They may be used in the treatment of bone and cartilage defects and in wound healing and related tissue repair.

This application is a continuation-in-part of Ser. Nos. 28,285 filedMar. 20, 1987 now abandoned; 943,332 filed Dec. 17, 1986 now abandoned;and 880,776 filed July 1, 1986 now abandoned. This application alsoclaims priority of PCT/US887/01537 filed June 30, 1987.

The present invention relates to a novel family of purified proteinsdesignated BMP-2 proteins and processes for obtaining them. Theseproteins may be used to induce bone and/or cartilage formation and inwound healing and tissue repair.

BMP-2 proteins are produced by culturing a cell transformed with a cDNAsubstantially as shown in Table II or Table III and recovering from theculture medium a protein containing substantially the 97 amino acidsequence #299 to #396 of Table II or amino acid #311 to #408 of TableIII.

Some members of the BMP-2 protein family are further characterized bythe ability of 200 nanograms of the BMP-2 protein to score at least +2in the Rosen-modified Sampath-Reddi assay of bone and/or cartilageformation.

BMP-2A is a member of the family of the BMP-2 proteins of the invention.We have previously referred to BMP-2A as BMP-2 or BMP-2 Class I. HumanBMP-2A (or hBMP-2A) is produced by culturing a cell transformed with acDNA substantially as shown in Table II and recovering from the culturemedium a protein containing the amino acid sequence of amino acid #299to amino acid #396 as shown in Table II. Human BMP-2A is furthercharacterized by the ability of 200 nanograms of the BMP-2A protein toscore at least +2 in the Rosen-modified Sampath - Reddi assay of boneand/or cartilage formation.

The bovine BMP-2A protein is a member of the family of BMP-2 proteins ofthe invention. It contains substantially the amino acid sequencerepresented by amino acid #32 to amino acid #129 of Table I. BovineBMP-2A is further characterized by the ability of 200 nanograms of thisprotein to score at least +2 in the Rosen-modified Sampath - Reddi assayof bone and/or cartilage formation.

Another member of the BMP-2 protein family is designated BMP-2B andwhich we have previously referred to as BMP-4 or BMP-2 Class II. BMP-2Bis produced by culturing a cell transformed with a cDNA substantially asshown in Table III and recovering from the culture medium a proteincontaining the amino acid sequence from amino acid #311 to #408 as shownin Table III. BMP-2B is further characterized by the ability of 200nanograms of this protein to score at least +2 in the Rosen-modifiedSampath - Reddi assay of bone and/or cartilage formation.

Another aspect of the invention provides pharmaceutical compositionscontaining a therapeutically effective amount of a BMP-2 protein in apharmaceutically acceptable vehicle or carrier. BMP-2 compositions mayalso be used for wound healing and tissue repair. The invention furtherprovides pharmaceutical compositions containing a therapeuticallyeffective amount of BMP-2A or BMP-2B in a pharmaceutically acceptablevehicle. Further compositions may contain both BMP-2A and BMP-2B in apharmaceutically acceptable vehicle. Compositions of the invention mayfurther include at least one other therapeutically useful agent such asthe BMP proteins BMP-1, and BMP-3 disclosed respectively in co-owned andconcurrently filed U.S. patent applications Ser. No. 179,101 and Ser.No. 179,197. Other therapeutically useful agents include growth factorssuch as epidermal growth factor (EGF), fibroblast growth factor (FGF),and transforming growth factor (TGF). The compositions may also includean appropriate matrix for instance, for supporting the composition andproviding a surface for bone and/or cartilage growth. The compositionsmay be employed in methods for treating a number of bone and/orcartilage defects, periodontal disease and various types of wounds.These methods, according to the invention, entail administering to apatient needing such bone and/or cartilage formation wound healing ortissue repair, an effective amount of a BMP-2 protein such as BMP-2Aand/or BMP-2B. These methods may also entail the administration of aprotein of the invention in conjunction with at least one of the novelBMP proteins disclosed in the co-owned applications described above. Inaddition, these methods may also include the administration of a BMP-2protein with other growth factors.

Still a further aspect of the invention are DNA sequences coding onexpression for a BMP-2 protein. Such sequences include the sequence ofnucleotides in a 5' to 3' direction illustrated in Tables I through IIIor DNA sequences which hybridize under stringent conditions with the DNAsequences of Tables I-III and encode a protein having the ability of 200nanograms of the protein to score at least +2 in the Rosen-modifiedSampath - Reddi assay of bone and/or cartilage formation described inExample III. Finally, allelic or other variations of the sequences ofTables I through III, whether such nucleotide changes result in changesin the peptide sequence or not, are also included in the presentinvention.

Still a further aspect of the invention is a vector containing a DNAsequence as described above in operative association with an expressioncontrol sequence therefor. Such vector may be employed in a novelprocess for producing a BMP-2 protein of the invention in which a cellline transformed with a DNA sequence encoding expression of a BMP-2protein in operative association with an expression control sequencetherefor, is cultured in a suitable culture medium and a BMP-2 proteinis isolated and purified therefrom. This claimed process may employ anumber of known cells both prokaryotic and eukaryotic as host cells forexpression of the polypeptide.

Other aspects and advantages of the present invention will be apparentupon consideration of the following detailed description and preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 comprises partial DNA sequence and derived amino acid sequence ofbovine BMP-2 (BMP-2A) from bacteriophage bP-21 ATCC #40310. FIG. 1corresponds to Table I further described below.

FIG. 2 comprises DNA sequence and derived amino acid sequence of humanBMP-2 (BMP-2A) from lambda U20S-39 ATCC #40345. FIG. 2 corresponds toTable II further described below.

FIG. 3 comprises DNA sequence and derived amino acid sequence of humanBMP-4 (BMP-2B) from lambda U20S-3 ATCC #40342. FIG. 3 corresponds toTable III further described below.

DETAILED DESCRIPTION OF THE INVENTION

The purified BMP-2 proteins of the present invention are produced byculturing a host cell transformed with a cDNA of Table II or III andrecovering from the culture medium a protein containing the 97 aminoacid sequence or a substantially homologous sequence as represented byamino acid #299 to #396 of Table II or #311 to #408 of Table III. SomeBMP-2 proteins are also characterized by the ability of 200 nanograms(ng) to score at least +2 in the Rosen-modified Sampath - Reddi assay ofbone and/or cartilage formation.

The BMP-2 proteins provided herein also include factors encoded by thesequences similar to those of Tables I-III, but into which modificationsare naturally provided (e.g. allelic variations in the nucleotidesequence which may result in amino acid changes in the polypeptide) ordeliberately engineered. For example, synthetic polypeptides may whollyor partially duplicate continuous sequences of the amino acid residuesof Tables I-III. These sequences, by virtue of sharing primary,secondary, or tertiary structural and conformational characteristicswith bone growth factor polypeptides of Tables I-III may possess bonegrowth factor biological properties in common therewith. Thus, they maybe employed as biologically active substitutes for naturally-occurringBMP-2A and BMP-2B and other BMP-2 polypeptides in therapeutic processes.

Other specific mutations of the sequences of BMP-2 proteins describedherein involve modifications of one or both of the glycosylation sites.The absence of glycosylation or only partial glycosylation results fromamino acid substitution or deletion at one or both of theasparagine-linked glycosylation recognition sites present in thesequences of BMP-2A and BMP-2B proteins shown in Tables I-III. Theasparagine-linked glycosylation recognition sites comprise tripeptidesequences which are specifically recognized by appropriate cellularglycosylation enzymes. These tripeptide sequences are eitherasparagine-X-threonine or asparagine-X-serine, where X is usually anyamino acid. A variety of amino acid substitutions or deletions at one orboth of the first or third amino acid positions of a glycosylationrecognition site (and/or amino acid deletion at the second position)results in non-glycosylation at the modified tripeptide sequence.

The present invention also encompasses the novel DNA sequences, free ofassociation with DNA sequences encoding other proteinaceous materials,and coding on expression for BMP-2 proteins such as BMP-2A and BMP-2B.These DNA sequences include those depicted in Tables I-III in a 5' to 3'direction and those sequences which hybridize under stringenthybridization conditions [see, T. Maniatis et al, Molecular Cloninq (ALaboratory Manual), Cold Spring Harbor Laboratory (1982), pages 387 to389]to the DNA sequences of Tables I-III.

Similarly, DNA sequences which code for BMP-2 proteins such as BMP-2Aand BMP-2B polypeptides coded for by the sequences of Tables I-III, butwhich differ in codon sequence due to the degeneracies of the geneticcode or allelic variations (naturally-occurring base changes in thespecies population which may or may not result in an amino acid change)also encode the novel factors described herein. Variations in the DNAsequences of Tables I-III which are caused by point mutations or byinduced modifications (including insertion, deletion, and substitution)to enhance the activity, half-life or production of the polypeptidesencoded thereby are also encompassed in the invention.

Another aspect of the present invention provides a novel method forproducing BMP-2 proteins. The method of the present invention involvesculturing a suitable cell line, which has been transformed with a DNAsequence coding on expression for a BMP-2 protein of the invention,under the control of known regulatory sequences. Suitable cells or celllines may be mammalian cells, such as Chinese hamster ovary cells (CHO).The selection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. See, e.g., Gething and Sambrook,Nature, 293:620-625 (1981), or alternatively, Kaufman et al, Mol. Cell.Biol., 5(7):1750-1759 (1985) or Howley et al, U.S. Pat. No. 4,419,446.Another suitable mammalian cell line, which is described in theaccompanying examples, is the monkey COS-1 cell line. The mammalian cellCV-1 may also be suitable.

Bacterial cells may also be suitable hosts. For example, the variousstrains of E. coli (e.g., HB101, MC1061) are well-known as host cells inthe field of biotechnology. Various strains of B. subtilis, Pseudomonas,other bacilli and the like may also be employed in this method.

Many strains of yeast cells known to those skilled in the art may alsobe available as host cells for expression of the polypeptides of thepresent invention. Additionally, where desired, insect cells may beutilized as host cells in the method of the present invention. See, e.g.Miller et al, Genetic Enqineering, 8:277-298 (Plenum Press 1986) andreferences cited therein.

Another aspect of the present invention provides vectors for use in themethod of expression of these novel BMP-2 and BMP-2 polypeptides.Preferably the vectors contain the full novel DNA sequences describedabove which code for the novel factors of the invention. Additionallythe vectors also contain appropriate expression control sequencespermitting expression of the BMP-2A protein sequences. Alternatively,vectors incorporating modified sequences as described above are alsoembodiments of the present invention and useful in the production of theBMP-2A and BMP-2B and other BMP-2B proteins. The vectors may be employedin the method of transforming cell lines and contain selected regulatorysequences in operative association with the DNA coding sequences of theinvention which are capable of directing the replication and expressionthereof in selected host cells. Useful regulatory sequences for suchvectors are known to one of skill in the art and may be selecteddepending upon the selected host cells. Such selection is routine anddoes not form part of the present invention.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage defects inhumans and other animals. Such a preparation employing a BMP-2 proteinsuch as BMP-2A and BMP-2B may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. A BMP-2 protein may be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A variety of osteogenic, cartilage-inducing and boneinducing factors have been described. See, e.g. European patentapplications 148,155 and 169,016 for discussions thereof.

The proteins of the invention may also be used in wound healing andrelated tissue repair. The types of wounds include, but are not limitedto burns, incisions and ulcers. (See, e.g. PCT Publication WO84/01106for discussion of wound healing and related tissue repair).

A further aspect of the invention is a therapeutic method andcomposition for repairing fractures and other conditions related tocartilage and/or bone defects or periodontal diseases. In addition, theinvention comprises therapeutic methods and compositions for woundhealing and tissue repair. Such compositions comprise a therapeuticallyeffective amount of at least one of the BMP-2 proteins of the inventionin admixture with a pharmaceutically acceptable vehicle, carrier ormatrix. It is expected that the proteins of the invention may act inconcert with or perhaps synergistically with other related proteins andgrowth factors. Further therapeutic methods and compositions of theinvention therefore comprise a therapeutic amount of at least one BMP-2protein of the invention with a therapeutic amount of at least one ofthe other BMP proteins disclosed in co-owned and concurrently filed U.S.applications described above. Further, BMP-2 proteins such as BMP-2A andBMP-2B may be combined with other agents beneficial to the treatment ofthe bone and/or cartilage defect, wound, or tissue in question. Theseagents include various growth factors such as epidermal growth factor(EGF), platelet derived growth factor (PDGF), transforming growth factor(TGF), and insulin-like growth factor (IGF). The preparation andformulation of such physiologically acceptable protein compositions,having due regard to pH, isotonicity, stability and the like, is withinthe skill of the art. The therapeutic compositions are also presentlyvaluable for veterinary applications due to the lack of speciesspecificity in BMP proteins. Particularly domestic animals andthoroughbred horses in addition to humans are desired patients for suchtreatment with BMP-2A and BMP-2B of the present invention.

BMP-2A may be used individually in a pharmaceutical composition. BMP-2Amay also be used in combination with BMP2B and/or one or more of theother BMP proteins disclosed in co-owned and co-pending US applicationsas discussed above.

BMP-2B may be used individually in pharmaceutical composition. Inaddition, it may be used in combination with other BMP proteins asdescribed above.

The therapeutic method includes administering the composition topically,systematically, or locally as an implant or device. When administered,the therapeutic composition for use in this invention is, of course, ina pyrogen-free, physiologically acceptable form. Further, thecomposition may desirably be encapsulated or injected in a viscous formfor delivery to the site of bone cartilage or tissue damage. Topicaladministration may be suitable for wound healing and tissue repair.Preferably for bone and/or cartilage formation, the composition wouldinclude a matrix capable of delivering BMP-2A, BMP-2B or other BMPprotein to the site of bone and/or cartilage damage, providing astructure for the developing bone and cartilage and optimally capable ofbeing resorbed into the body. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the BMP-2compositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are nonbiodegradable and chemicallydefined, such as sintered hydroxyapatite, bioglass, aluminates, or otherceramics. Matrices may be comprised of combinations of any of the abovementioned types of material, such as polylactic acid and hydroxyapatiteor collagen and tricalciumphosphate. The bioceramics may be altered incomposition, such as in calcium-aluminate-phosphate and processing toalter pore size, particle size, particle shape, and biodegradability.

The dosage regimen will be determined by the attending physicianconsidering various factors which modify the action of the BMP-2protein, e.g. amount of bone weight desired to be formed, the site ofbone damage, the condition of the damaged bone, the size of a wound,type of damaged tissue, the patient's age, sex, and diet, the severityof any infection, time of administration and other clinical factors. Thedosage may vary with the type of matrix used in the reconstitution andthe type of BMP in the composition of BMP's. The addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of bone growth and/or repair, e.g. x-rays.

The following examples illustrate practice of the present invention inrecovering and characterizing bovine BMP-2A protein and employing it torecover the human proteins BMP-2A and BMP-2B, obtaining the humanproteins and in expressing the proteins via recombinant techniques.

EXAMPLE I Isolation of Bovine Bone Inductive Factor

Ground bovine bone powder (20-120 mesh, Helitrex) is prepared accordingto the procedures of M. R. Urist et al., Proc. Natl Acad. Sci USA,70:3511 (1973) with elimination of some extraction steps as identifiedbelow. Ten kgs of the ground powder is demineralized in successivechanges of 0.6N HCl at 4° C. over a 48 hour period with vigorousstirring. The resulting suspension is extracted for 16 hours at 4° C.with 50 liters of 2M CaCl₂ and 10 mM ethylenediamine-tetraacetic acid[EDTA], and followed by extraction for 4 hours in 50 liters of 0.5MEDTA. The residue is washed three times with distilled water before itsresuspension in 20 liters of 4M guanidine hydrochloride [GuCl], 20mMTris (pH 7.4), 1 mM N-ethylmaleimide, 1 mM iodoacetamide, b 1 mMphenylmethylsulfonyl fluorine as described in Clin. Orthop. Rel. Res.,171: 213 (1982). After 16 to 20 hours the supernatant is removed andreplaced with another 10 liters of GuCl buffer. The residue is extractedfor another 24 hours.

The crude GuCl extracts are combined, concentrated approximately 20times on a Pellicon apparatus with a 10,000 molecular weight cut-offmembrane, and then dialyzed in 50mM Tris, 0.1 M NaCl, 6M urea (pH7.2),the starting buffer for the first column. After extensive dialysis theprotein is loaded on a 4 liter DEAE cellulose column and the unboundfractions are collected.

The unbound fractions are concentrated and dialyzed against 50mM NaAc,50mM NaCl (pH 4.6) in 6M urea. The unbound fractions are applied to acarboxymethyl cellulose column. Protein not bound to the column isremoved by extensive washing with starting buffer, and the materialcontaining protein having bone and/or cartilage formation activity asmeasured by the Rosen-modified Sampath - Reddi assay (described inExample III below) desorbed from the column by 50mM NaAc, 0.25mM NaCl,6M urea (pH 4.6). The protein from this step elution is concentrated 20-to 40- fold, then diluted 5 times with 80mM KPO₄, 6M urea (pH6.0). ThepH of the solution is adjusted to 6.0 with 500mM K₂ HPO₄. The sample isapplied to an hydroxylapatite column (LKB) equilibrated in 80mM KPO₄, 6Murea (pH6.0) and all unbound protein is removed by washing the columnwith the same buffer. Protein having bone and/or cartilage formationactivity is eluted with 100mM KPO₄ (pH7.4) and 6M urea.

The protein is concentrated approximately 10 times, and solid NaCl addedto a final concentration of 0.15M. This material is applied to aheparin - Sepharose column equilibrated in 50mM KPO₄, 150mM NaCl, 6Murea (pH7.4). After extensive washing of the column with startingbuffer, a protein with bone and/or cartilage inductive activity iseluted by 50mM KPO₄, 700mM NaCl, 6M urea (pH7.4). This fraction isconcentrated to a minimum volume, and 0.4ml aliquots are applied toSuperose 6 and Superose 12 columns connected in series, equilibratedwith 4M GuCl, 20mM Tris (pH7.2) and the columns developed at a flow rateof 0.25ml/min. The protein demonstrating bone and/or cartilage inductiveactivity has a relative migration on SDS-PAGE corresponding toapproximately 30,000 dalton protein.

The above fractions from the superose columns are pooled, dialyzedagainst 50mM NaAc, 6M urea (pH4.6), and applied to a Pharmacia MonoS HRcolumn. The column is developed with a gradient to 1.0 M NaCl, 50mMNaAc, 6M urea (pH4.6). Active bone and/or cartilage formation fractionsare pooled and brought to pH3.0 with 10% trifluoroacetic acid (TFA). Thematerial is applied to a 0.46×25 cm Vydac C4 column in 0.1% TFA and thecolumn developed with a gradient to 90% acetonitrile, 0.1% TFA (31.5%acetonitrile, 0.1% TFA to 49.5% acetonitrile, 0.1% TFA in 60 minutes at1 ml per minute). Active material is eluted at approximately 40-44%acetonitrile. Aliquots of the appropriate active fractions are iodinatedby one of the following methods: P. J. McConahey et al, Int. Arch.Allerov, 29:185-189 (1966); A. E. Bolton et al, Biochem J., 133:529(1973) ; and D. F. Bowen-Pope, J. Biol. Chem., 237:5161 (1982). Theiodinated proteins present in these fractions are analyzed by SDS gelelectrophoresis and urea Triton×100 isoelectric focusing. At this stage,the protein having bone and/or cartilage forming activity is estimatedto be approximately 10-50% pure.

EXAMPLE II Characterization of Bovine Bone Inductive Factor A. MolecularWeight

Approximately 20 ug protein from Example I is lyophilized andredissolved in 1×SDS sample buffer. After 15 minutes of heating at 37°C., the sample is applied to a 15% SDS polyacrylamide gel and thenelectrophoresed with cooling. The molecular weight is determinedrelative to prestained molecular weight standards (Bethesda ResearchLabs). Immediately after completion, the gel lane containing bone and/orcartilage forming material is sliced into 0.3cm pieces. Each piece ismashed and 1.4ml of 0.1% SDS is added. The samples are shaken gentlyovernight at room temperature to elute the protein. Each gel slice isdesalted to prevent interference in the biological assay. Thesupernatant from each sample is acidified to pH 3.0 with 10% TFA,filtered through a 0.45 micron membrane and loaded on a 0.46cm×5cm C4Vydac column developed with a gradient of 0.1% TFA to 0.1% TFA, 90% CH₃CN. The appropriate bone and/or cartilage inductive protein-containingfractions are pooled and reconstituted with 20 mg rat matrix andassayed. In this gel system, the majority of bone and/or cartilageinductive fractions have the mobility of a protein having a molecularweight of approximately 28,000-30,000 daltons.

B. Isoelectric Focusing

The isoelectric point of bone inductive factor activity is determined ina denaturing isoelectric focusing system. The Triton X100 urea gelsystem (Hoeffer Scientific) is modified as follows: 1) 40% of theampholytes used are Servalyte 3/10; 60% are Servalyte 7-9; and 2) thecatholyte used is 40 mM NaOH. Approximately 20 ug of protein fromExample I is lyophilized, dissolved in sample buffer and applied to theisoelectrofocusing gel. The gel is run at 20 watts, 10° C. forapproximately 3 hours. At completion the lane containing bone and/orcartilage inductive factor is sliced into 0.5 cm slices. Each piece ismashed in 1.0 ml 6M urea, 5 mM Tris (pH 7.8) and the samples agitated atroom temperature. The samples are acidified, filtered, desalted andassayed as described above. The major portion of activity as determinedby the Rosen-modified Sampath - Reddi assay migrates in a mannerconsistent with a pI of about 8.8-9.2.

C. Subunit Characterization

The subunit composition of the isolated bovine bone protein is alsodetermined. Pure bone inductive factor is isolated from a preparative15% SDS gel as described above. A portion of the sample is then reducedwith 5 mM DTT in sample buffer and re-electrophoresed on a 15% SDS gel.The approximately 28-30 kd protein yields two major bands atapproximately 18-20 kd and approximately 16-18 kd, as well as a minorband at approximately 28-30 kd. The broadness of the two bands indicatesheterogeneity caused most probably by glycosylation, other posttranslational modification, proteolytic degradation or carbamylation.

EXAMPLE III Rosen Modified Sampath-Reddi Assay

A modified version of the rat bone formation assay described in Sampathand Reddi, Proc. Natl. Acad. Sci. U.S.A., 80:6591-6595 (1983) is used toevaluate bone and/or cartilage activity of the bovine protein obtainedin Example I and the BMP-2 proteins of the invention. This modifiedassay is herein called the Rosen-modified Sampath-Reddi assay. Theethanol precipitation step of the Sampath-Reddi procedure is replaced bydialyzing (if the composition is a solution) or diafiltering (if thecomposition is a suspension) the fraction to be assayed against water.The solution or suspension is then redissolved in 0.1% TFA, and theresulting solution added to 20 mg of rat matrix. A mock rat matrixsample not treated with the protein serves as a control. This materialis frozen and lyophilized and the resulting powder enclosed in #5gelatin capsules. The capsules are implanted subcutaneously in theabdominal thoracic area of 21-49 day old male Long Evans rats. Theimplants are removed after 7-14 days. Half of each implant is used foralkaline phosphatase analysis [See, A. H. Reddi et al., Proc. Natl AcadSci., 69:1601 (1972)].

The other half of each implant is fixed and processed for histologicalanalysis. About 1 um glycolmethacrylate sections are stained with VonKossa and acid fuschin to score the amount of induced bone and cartilageformation present in each implant. The terms +1 through +5 represent thearea of each histological section of an implant occupied by new boneand/or cartilage cells and matrix. A score of +5 indicates that greaterthan 50% of the implant is new bone and/or cartilage produced as adirect result of protein in the implant. A score of +4, +3, +2 and +1would indicate that greater than 40%, 30%, 20% and 10% respectively ofthe implant contains new cartilage and/or bone.

The rat matrix samples containing at least 200 ng of protein obtained inExample I result in bone and/or cartilage formation that filled morethan 20% of the implant areas that was sectioned for histology. Thisprotein therefore scores at least +2 in the Rosen-modified Sampath-Reddiassay. The dose response of the matrix samples indicates that the amountof bone and/or cartilage formed increases with the amount of protein inthe sample. The control sample did not result in any bone and/orcartilage formation. The purity of the protein assayed is approximately10-15% pure.

The bone and/or cartilage formed is physically confined to the spaceoccupied by the matrix. Samples are also analyzed by SDS gelelectrophoresis and isoelectric focusing as described above, followed byautoradiography. Analysis reveals a correlation of activity with proteinbands at 28-30 kd and a pI of approximately 8.8-9.2. To estimate thepurity of the protein in a particular fraction an extinction coefficientof 1 OD/mg-cm is used as an estimate for protein and the protein is runon SDS PAGE followed by silver straining or radioiodination andautoradiography.

EXAMPLE IV Bovine BMP-2A

The protein composition of Example IIA of molecular weight 28-30 kd isreduced as described in Example IIC and digested with trypsin. Eighttryptic fragments are isolated by standard procedures having thefollowing amino acid sequences: ##STR1##

Two probes consisting of pools of oligonucleotides (or uniqueoligonucleotides) are designed according to the method of R. Lathe, J.Mol. Biol., 183(1):1-12 (1985) on the basis of the amino acid sequenceof Fragment 3 and synthesized on an automated DNA synthesizer asdescribed above.

    Probe #1: A C N A C C A T [A/G] T C [T/C] T G [A/G] A T

    Probe #2: C A [A/G] G A [T/C] A T G G T N G T N G A

Because the genetic code is degenerate (more than one codon can code forthe same amino acid), the number of oligonucleotides in a probe pool isreduced based on the frequency of codon usage in eukaryotes, therelative stability of G:T base pairs, and the relative infrequency ofthe dinucleotide CpG in eukaryotic coding sequences [See J. J. Toole etal, Nature. 312:342-347 (1984)]. Bracketed nucleotides are alternatives."N" means either A, T, C or G. These probes are radioactively labeledand employed to screen a bovine genomic library. The library isconstructed as follows: Bovine liver DNA is partially digested with therestriction endonuclease enzyme Sau 3A and sedimented through a sucrosegradient. Size fractionated DNA in the range of 15-30 kb is then ligatedto the vector lambda J' Bam H1 arms [Mullins et al., Nature, 08:856-858(1984)]. The library is plated at 8000 recombinants per plate. Duplicatenitrocellulose replicas of the plaques are made and amplified accordingto a modification of the procedure of Woo et al, Proc. Natl. Acad. Sci.USA, 75:368-891 (1978). Probe #1 is hybridized to the set of filters in3M tetramethylammonium chloride (TMAC), 0.1M sodium phosphate pH6.5, 1mM EDTA, 5×Denhardts, 0.6% SDS, 100 ug/ml salmon sperm DNA at 48 degreesC., and washed in 3M TMAC, 50 mM Tris pH8.0 at 50 degrees C. Theseconditions minimize the detection of mismatches to the 17 mer probe pool[see, Wood et al, Proc. Natl. Acad. Sci, U.S.A., 82:1585-1588 (1985)].

400,000 recombinants are screened by this procedure. One duplicatepositive is plaque purified and the DNA is isolated from a plate lysateof the recombinant bacteriophage designated lambda bP-21. BacteriophagebP-21 was deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville, Maryland USA (hereinafter the "ATCC") underaccession number ATCC 40310 on Mar. 6, 1987. This deposit as well as theother deposits contained herein meets the requirements of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure and Regulations thereunder. ThebP-21 clone encodes at least a portion of a bovine BMP-2 proteindesignated bovine BMP-2A or bBMP-2A.

The oligonucleotide hybridizing region of this BMP-2A clone is localizedto an approximately 1.2 kb Sac I restriction fragment which is subclonedinto M13 and sequenced by standard techniques. The partial DNA sequenceand derived amino acid sequence of this Sac I fragment and thecontiguous Hind III-Sac I restriction fragment of bP-21 are shown belowin Table I. The BMP-2A peptide sequence from this clone is 129 aminoacids in length and is encoded by the DNA sequence from nucleotide #1through nucleotide #387. The amino acid sequence corresponding to thetryptic fragment isolated from the bovine bone 28 to 30 kd material isunderlined in Table I. The underlined portion of the sequencecorresponds to tryptic Fragment 3 above from which the oligonucleotideprobes for BMP-2A are designed. The predicted amino acid sequenceindicates that tryptic Fragment 3 is preceded by a basic residue (K) asexpected considering the specificity of trypsin. The arginine residueencoded by the CGT triplet is presumed to be the carboxy-terminus of theprotein based on the presence of a stop codon (TAG) adjacent to it.

                  TABLE I                                                         ______________________________________                                         ##STR2##                                                                      ##STR3##                                                                      ##STR4##                                                                      ##STR5##                                                                      ##STR6##                                                                      ##STR7##                                                                      ##STR8##                                                                      ##STR9##                                                                      ##STR10##                                                                     ##STR11##                                                                     ##STR12##                                                                     ##STR13##                                                                     ##STR14##                                                                     ##STR15##                                                                     ##STR16##                                                                     ##STR17##                                                                     ##STR18##                                                                     ##STR19##                                                                     ##STR20##                                                                    ______________________________________                                    

EXAMPLE V Human BMP-2A and BMP-2B

The HindIII-SacI bovine genomic BMP-2A fragment described in Example IVis subcloned into an M13 vector. A ³² P-labeled single-stranded DNAprobe is made from a template preparation of this subclone. This probeis used to screen polyadenylated RNAs from various cell and tissuesources. Polyadenylated RNAs from various cell and tissue sources areelectrophoresed on formaldehyde-agarose gels and transferred tonitrocellulose by the method of Toole et al., supra. The probe is thenhybridized to the nitrocellulose blot in 50% formamide, 5×SSC, 0.1% SDS,40 mM sodium phosphate pH 6.5, 100 ug/ml denatured salmon sperm DNA, and5 mM vanadyl ribonucleosides at 42° C. overnight and washed at 65° C. in0.2×SSC, 0.1% SDS. A hybridizing band corresponding to an mRNA speciesof approximately 3.8 kb is detected in the lane containing RNA from thehuman cell line U-2 OS. The HindIII-SacI fragment is labeled with ³² Pby nick translation and used to screen the nitrocellulose filterreplicas of the above-described U-2 OS cDNA library by hybridization instandard hybridization buffer at 65° overnight followed by washing in 1×SSC, 0.1% SDS at 65°. Twelve duplicate positive clones are picked andreplated for secondaries. Duplicate nitrocellulose replicas are made ofthe secondary plates and both sets hybridized to the bovine genomicprobe as the primary screening was performed. One set of filters is thenwashed in 1×SSC, 0.1% SDS; the other in 0.1×SSC, 0.1% SDS at 65°.

Two classes of hBMP-2 cDNA clones are evident based on strong (4recombinants) or weak (7 recombinants) hybridization signals under themore stringent washing conditions (0.1×SSC, 0.1% SDS). All 11recombinant bacteriophage are plaque purified, small scale DNApreparations made from plate lysates of each, and the inserts subclonedinto pSP65 and into M13 for sequence analysis. Sequence analysis of thestrongly hybridizing clones designated hBMP-2A (previously designatedBMP-2 and BMP-2 Class I) indicates that they have extensive sequencehomology with the sequence given in Table I. These clones are thereforecDNA encoding the human equivalent of the protein encoded by the bBMP-2Agene whose partial sequence is given in Table I. Sequence analysis ofthe weakly hybridizing recombinants designated hBMP-2B (previouslydesignated BMP-4 and BMP-2 Class II) indicates that they are also quitehomologous with the sequence given in Table I at the 3' end of theircoding regions, but less so in the more 5' regions. Thus they encode ahuman protein of similar, though not identical, structure to that above.

Full length human BMP-2A cDNA clones are obtained in the followingmanner. The 1.5 kb insert of one of the BMP-2B subclones (II-10-1) isisolated and radioactively labeled by nick-translation. One set of thenitrocellulose replicas of the U-2 OS cDNA library screened above (50filters, corresponding to 1,000,000 recombinant bacteriophage) arerehybridized with this probe under stringent conditions (hybridizationat 65° in standard hybridization buffer; washing at 65° in 0.2×SSC, 0.1%SDS). All recombinants which hybridize to the bovine genomic probe whichdo not hybridize to the BMP-2B probe are picked and plaque purified (10recombinants). Plate stocks are made and small scale bacteriophage DNApreparations made. After subcloning into M13, sequence analysisindicates that 4 of these represent clones which overlap the originalBMP-2A clone. One of these, lambda U20S-39, contains an approximately1.5 kb insert and was deposited with the ATCC on Jun. 16, 1987 underaccession number 40345. The partial DNA sequence (compiled from lambdaU20S-39 and several other hBMP-2A cDNA recombinants) and derived aminoacid sequence are shown below in Table II. Lambda U20S-39 is expected tocontain all of the nucleotide sequence necessary to encode the entirehuman counterpart of the protein BMP-2A encoded by the bovine genesegment whose partial sequence is presented in Table I. The BMP-2Aprotein encoded by Table II is contemplated to contain the 97 amino acidsequence from amino acid #299 to #396 or a sequence substantiallyhomologous thereto. This human cDNA hBMP-2A contains an open readingframe of 1188 bp, encoding a protein of 396 amino acids. The protein ispreceded by a 5' untranslated region of 342 bp with stop codons in allframes. The 13 bp region preceding this 5' untranslated regionrepresents a linker used in the cDNA cloning procedure. This protein of396 amino acids has a molecular weight of 45 kd based on this amino acidsequence. It is contemplated that this sequence represents the primarytranslation product. It is further contemplated that BMP-2A maycorrespond to the approximately 18-20 kd subunit of Example IIC. Thesequence corresponding to the sequence tryptic Fragment 3 of Example IVis underlined in Table II.

                  TABLE II                                                        ______________________________________                                         ##STR21##                                                                     ##STR22##                                                                     ##STR23##                                                                     ##STR24##                                                                     ##STR25##                                                                     ##STR26##                                                                     ##STR27##                                                                     ##STR28##                                                                     ##STR29##                                                                     ##STR30##                                                                     ##STR31##                                                                     ##STR32##                                                                     ##STR33##                                                                     ##STR34##                                                                     ##STR35##                                                                     ##STR36##                                                                     ##STR37##                                                                     ##STR38##                                                                     ##STR39##                                                                     ##STR40##                                                                     ##STR41##                                                                     ##STR42##                                                                     ##STR43##                                                                     ##STR44##                                                                     ##STR45##                                                                     ##STR46##                                                                     ##STR47##                                                                     ##STR48##                                                                     ##STR49##                                                                     ##STR50##                                                                     ##STR51##                                                                     ##STR52##                                                                     ##STR53##                                                                     ##STR54##                                                                     ##STR55##                                                                     ##STR56##                                                                     ##STR57##                                                                     ##STR58##                                                                     ##STR59##                                                                     ##STR60##                                                                     ##STR61##                                                                     ##STR62##                                                                     ##STR63##                                                                     ##STR64##                                                                     ##STR65##                                                                     ##STR66##                                                                     ##STR67##                                                                     ##STR68##                                                                     ##STR69##                                                                     ##STR70##                                                                     ##STR71##                                                                     ##STR72##                                                                    ______________________________________                                    

Full-length BMP-2B human cDNA clones are obtained in the followingmanner. The 200 bp EcoRI-SacI fragment from the 5' end of the BMP-2Brecombinant II-10-1 is isolated from its plasmid subclone, labeled bynick-translation, and hybridized to a set of duplicate nitrocellulosereplicas of the U-2 OS cDNA library (25 filters/set; representing500,000 recombinants). Hybridization and washing are performed understringent conditions as described above. 16 duplicate positives arepicked and replated for secondaries. Nitrocellulose filter replicas ofthe secondary plates are made and hybridized to an oligonucleotide whichwas synthesized to correspond to the sequence of II-10-1 and is of thefollowing sequence:

    CGGGCGCTCAGGATACTCAAGACCAGTGCTG

Hybridization is in standard hybridization buffer AT 50° C. with washingat 50° in 1×SSC, 0.1% SDS. 14 recombinant bacteriophage which hybridizeto this oligonucleotide are plaque purified. Plate stocks are made andsmall scale bacteriophage DNA preparations made. After sucloning 3 ofthese into M13, sequence analysis indicates that they represent cloneswhich overlap the original BMP-2B clone. One of these, lambda U20S-3,was deposited with the ATCC under accession number 40342 on Jun. 16,1987. U20S-3 contains an insert of approximately 1.8 kb. The partial DNAsequence and derived amino acid sequence of U20S-3 are shown below inTable III. This clone is expected to contain all of the nucleotidesequence necessary to encode the entire human BMP-2B protein. The BMP-2Bprotein encoded by Table III is contemplated to contain the 97 aminoacid sequence from amino acid #311 to #408 or a sequence substantiallyhomologous thereto. This cDNA contains an open reading frame of 1224 bp,encoding a protein of 408 amino acids, preceded by a 5' untranslatedregion of 394 bp with stop codons in all frames, and contains a 3'untranslated region of 308 bp following the in-frame stop codon. The 8bp region preceding the 5' untranslated region represents a linker usedin the cDNA cloning procedure. This protein of 408 amino acids hasmolecular weight of 47 kd and is contemplated to represent the primarytranslation product. A sequence similar though not identical to trypticFragment 3 of Example IV is underlined in Table III.

                  TABLE III                                                       ______________________________________                                         ##STR73##                                                                     ##STR74##                                                                     ##STR75##                                                                     ##STR76##                                                                     ##STR77##                                                                     ##STR78##                                                                     ##STR79##                                                                     ##STR80##                                                                     ##STR81##                                                                     ##STR82##                                                                     ##STR83##                                                                     ##STR84##                                                                     ##STR85##                                                                     ##STR86##                                                                     ##STR87##                                                                     ##STR88##                                                                     ##STR89##                                                                     ##STR90##                                                                     ##STR91##                                                                     ##STR92##                                                                     ##STR93##                                                                     ##STR94##                                                                     ##STR95##                                                                     ##STR96##                                                                     ##STR97##                                                                     ##STR98##                                                                     ##STR99##                                                                     ##STR100##                                                                    ##STR101##                                                                    ##STR102##                                                                    ##STR103##                                                                    ##STR104##                                                                    ##STR105##                                                                    ##STR106##                                                                    ##STR107##                                                                    ##STR108##                                                                    ##STR109##                                                                    ##STR110##                                                                    ##STR111##                                                                    ##STR112##                                                                    ##STR113##                                                                    ##STR114##                                                                    ##STR115##                                                                    ##STR116##                                                                    ##STR117##                                                                    ##STR118##                                                                    ##STR119##                                                                    ##STR120##                                                                    ##STR121##                                                                    ##STR122##                                                                    ##STR123##                                                                    ##STR124##                                                                    ##STR125##                                                                    ##STR126##                                                                    ##STR127##                                                                    ##STR128##                                                                    ##STR129##                                                                    ##STR130##                                                                    ##STR131##                                                                    ##STR132##                                                                    ##STR133##                                                                    ##STR134##                                                                    ##STR135##                                                                    ##STR136##                                                                   ______________________________________                                    

The sequences of BMP-2A and BMP-2B, as shown in Tables II and III, havesignificant homology to the beta (B) and beta (A) subunits of theinhibins. The inhibins are a family of hormones which are presentlybeing investigated for use in contraception. See, A. J. Mason et al,Nature, 318:659-663 (1985). To a lesser extent they are also homologousto Mullerian inhibiting substance (MIS), a testicular glycoprotein thatcauses regression of the Mullerian duct during development of the maleembryo and transforming growth factor-beta (TGF-b) which can inhibit orstimulate growth of cells or cause them to differentiate. Furthermore,the sequences of Tables II and III indicate that BMP-2A and 2B havesignificant homology to the Drosophila decapentaplegic (DPP-C) locustranscript. See, J. Massague, Cell, 49:437-438 (1987); R. W. Padgett etal, Nature, 325:81-84 (1987); R. L. Cate et al, Cell 45: 685-698 (1986).It is considered possible therefore that a BMP-2 protein is the humanhomolog of the protein made from this transcript from this developmentalmutant locus. BMP-2A and BMP-2B share sequence similarity with Vgl. VglmRNA has been localized to the vegetal hemisphere of Xenopus oocytes.During early development, it is distributed throughout the endoderm, butthe mRNA is not detectable after blastula formation has occurred. TheVgl protein may be the signal used by the endorderm cells to commitectodermal cells to become the embryonic mesoderm.

EXAMPLE VI Expression of BMP-2A and BMP-2B

In order to produce bovine, human or other mammalian BMP-2 proteins, theDNA encoding it is transferred into an appropriate expression vector andintroduced into mammalian cells or other preferred eukaryotic orprokaryotic hosts by conventional genetic engineering techniques. Thepresently preferred expression system for biologically activerecombinant human BMP-2A and BMP-2B is stably transformed mammaliancells.

One skilled in the art can construct mammalian expression vectors byemploying the sequence of Tables I-III or other modified sequences andknown vectors, such as pCD [Okayama et al., Mol. Cell Biol., 2:161-170(1982)]and pJL3, pJL4 [Gough et al., EMBO J., 4:645-653 (1985)]. Thetransformation of these vectors into appropriate host cells can resultin expression of BMP-2A or BMP-2B. One skilled in the art couldmanipulate the sequences of Tables I-III by eliminating or replacing themammalian regulatory sequences flanking the coding sequence withbacterial sequences to create bacterial vectors for intracellular orextracellular expression by bacterial cells. For example, the codingsequences could be further manipulated (e.g. ligated to other knownlinkers or modified by deleting non-coding sequences there-from oraltering nucleotides therein by other known techniques). The modifiedBMP-2A or BMP-2B coding sequence could then be inserted into a knownbacterial vector using procedures such as described in T. Taniguchi etal., Proc. Natl Acad. Sci. USA, 77:5230-5233 (1980). This exemplarybacterial vector could then be transformed into bacterial host cells anda BMP-2 protein expressed thereby. For a strategy for producingextracellular expression of a BMP-2 protein in bacterial cells., see,e.g. European patent application EPA 177,343.

Similar manipulations can be performed for the construction of an insectvector [See, e.g. procedures described in published European patentapplication 155,476] for expression in insect cells. A yeast vectorcould also be constructed employing yeast regulatory sequences forintracellular or extracellular expression of the factors of the presentinvention by yeast cells. [See, e.g., procedures described in publishedPCT application WO86/00639 and European patent application EPA 123,289].

A method for producing high levels of a BMP-2 protein of the inventionfrom mammalian cells involves the construction of cells containingmultiple copies of the heterologous BMP2 gene. The heterologous gene canbe linked to an amplifiable marker, e.g. the dihydrofolate reductase(DHFR) gene for which cells containing increased gene copies can beselected for propagation in increasing concentrations of methotrexate(MTX) according to the procedures of Kaufman and Sharp, J. Mol. Biol.,159:601-629 (1982). This approach can be employed with a number ofdifferent cell types. For example, a plasmid containing a DNA sequencefor a BMP-2A or BMP-2B of the invention in operative association withother plasmid sequences enabling expression thereof and the DHFRexpression plasmid pAdA26SV(A)3 [Kaufman and Sharp, Mol. Cell. Biol.,2:1304 (1982)]can be co-introduced into DHFR-deficient CHO cells,DUKX-BII, by calcium phosphate coprecipitation and transfection,electroperation or protoplast fusion. DHFR expressing transformants areselected for growth in alpha media with dialyzed fetal calf serum, andsubsequently selected for amplification by growth in increasingconcentrations of MTX (sequential steps in 0.02, 0.2, 1.0 and 5 uM MTX)as described in Kaufman et al., Mol Cell Biol., 5:1750 (1983).Transformants are cloned, and biologically active BMP-2A or BMP-2Bexpression is monitored by the Rosen-modified Sampath - Reddi rat boneformation assay described above in Example III. BMP-2A and BMP-2Bexpression should increase with increasing levels of MTX resistance.Similar procedures can be followed to produce other related BMP-2proteins.

As one specific example, to produce the BMP-2A or BMP-2B of Example V,the insert of U20S-39 or U20S respectively, is released from the vectorarms by digestion with ECORI and subcloned into the mammalian expressionvector pMT2CX digested with ECORI. Plasmid DNA from this subclone istransfected into COS cells by the DEAE-dextran procedure [Sompayrac andDanna PNAS 78:7575-7578 (1981); Luthman and Magnusson, Nucl.Acids Res.11: 1295-1308 (1983)] and the cells are cultured. Serum-free 24 hr.conditioned medium supernatant is collected from the cells starting40-70 hr. post-transfection.

The mammalian expression vector pMT2 Cla-Xho (pMT2 CX) is a derivativeof p91023 (b) (Wong et al., Science 228:810-815, 1985) differing fromthe latter in that it contains the ampicillin resistance gene in placeof the tetracycline resistance gene and further contains a XhoI site forinsertion of cDNA clones. The functional elements of pMT2 Cla-Xho havebeen described (Kaufman, R. J., 1985, Proc. Natl. Acad. Sci. USA82:689-693) and include the adenovirus VA genes, the SV40 origin ofreplication including the 72 bp enhancer, the adenovirus major latepromoter including a 5' splice site and the majority of the adenovirustripartite leader sequence present on adenovirus late mRNAs, a 3' spliceacceptor site, a DHFR insert, the SV40 early polyadenylation site(SV40), and pBR322 sequences needed for propagation in E. coli.

Plasmid pMT2 Cla-Xho is obtained by EcoRI digestion of pMT2-VWF, whichhas been deposited with the American Type Culture Collection (ATCC),Rockville, MD (USA) under accession number ATCC 67122. EcoRI digestionexcises the cDNA insert present in pMT2-VWF, yielding pMT2 in linearform which can be ligated and used to transform E. coli HB 101 or DH-5to ampicillin resistance. Plasmid pMT2 DNA can be prepared byconventional methods. pMT2CX is then constructed by digesting pMT2 withEco RV and XbaI, treating the digested DNA with Klenow fragment of DNApolymerase I, and ligating Cla linkers (NEBiolabs, CATCGATG). Thisremoves bases 2266 to 2421 starting from the Hind III site near the SV40origin of replication and enhancer sequences of pMT2. Plasmid DNA isthen digested with EcoRI, blunted as above, and ligated to an EcoRIadapter,

    5'PO.sub.4 -AATTCCTCGAGAGCT 3'

    3'GGAGCTCTCGA 5'

digested with XhoI, and ligated, yielding pMT2 Cla-Xho, which may thenbe used to transform E. coli to ampicillin resistance. Plasmid pMT2Cla-Xho DNA may be prepared by conventional methods.

Example VII Biological Activity of Expressed BMP-2A and BMP-2B

To measure the biological activity of the expressed BMP-2A and BMP-2Bobtained in Example VI above, the protein is partially purified on aHeparin Sepharose column. 4 ml of the collected post transfectionconditioned medium supernatant from one 100 mm culture dish isconcentrated approximately 10 fold by ultrafiltration on a YM 10membrane and then dialyzed against 20mM Tris, 0.15 M NaCl, pH 7.4(starting buffer). This material is then applied to a 1.1 ml HeparinSepharose column in starting buffer. Unbound proteins are removed by an8 ml wash of starting buffer, and bound proteins, including BMP-2proteins, are desorbed by a 3-4 ml wash of 20 mM Tris, 2.0 M NaCl, pH7.4.

The proteins bound by the Heparin column are concentrated approximately10-fold on a Centricon 10 and the salt reduced by diafiltration with0.1% trifluoroacetic acid. Purified BMP-2 proteins are approximately 95%substantially free from other proteinaceous materials. The appropriateamount of this solution is mixed with 20 mg of rat matrix and thenassayed for in vivo bone and/or cartilage formation activity by theRosen-modified Sampath - Reddi assay. A mock transfection supernatantfractionation is used as a control.

The implants containing rat matrix to which specific amounts of humanBMP-2A or BMP-2B have been added are removed from rats after seven daysand processed for histological evaluation. Representative sections fromeach implant are stained for the presence of new bone mineral with vonKossa and acid fuschin, and for the presence of cartilage-specificmatrix formation using toluidine blue. The types of cells present withinthe section, as well as the extent to which these cells displayphenotype are evaluated and scored as described in Example III.

Addition of human BMP-2A or BMP-2B to the matrix material resulted information of cartilage-like nodules at 7 days post implantation. Thechondroblast-type cells were recognizable by shape and expression ofmetachromatic matrix. The assay results indicate that approximately 200ng of BMP-2A or BMP-2B results on a score of at least +2. The amount ofactivity observed for human BMP-2A or BMP-2B indicates that it may bedependent upon the amount of human BMP-2A or BMP-2B protein added to thematrix sample.

Similar levels of activity are seen in the Heparin Sepharosefractionated COS cell extracts. Partial purification is accomplished ina similar manner as described above except that 6 M urea is included inall the buffers.

The procedures described above may be employed to isolate other relatedBMP-2 proteins of interest by utilizing the bovine BMP-2A and BMP-2Bproteins as a probe source. Such other BMP-2 proteins may find similarutility in, inter alia, fracture repair, wound healing and tissuerepair.

The foregoing descriptions detail presently preferred embodiments of thepresent invention. Numerous modifications and variations in practicethereof are expected to occur to those skilled in the art uponconsideration of these descriptions. Those modifications and variationsare believed to be encompassed within the claims appended hereto.

What is claimed is:
 1. An isolated DNA sequence encoding anosteoinductive protein said DNA sequence comprising a coding sequenceselected from the group consisting of(a) nucleotide #1 throughnucleotide #387 of figure I, (b) nucleotide #356 through nucleotide#1543 of figure II, (c) Nucleotide #402 through nucleotide #1626 offigure III, (d) naturally occurring allelic sequences and equivalentdegenerative codon sequences of (a), (b), and (c); and (e) sequenceswhich(1) hybridize to any of sequences (a), (b), (c), or (d) understringent hybridization conditions; and (2) encode a proteincharacterized by the ability to induce the formation of bone and/orcartilage.
 2. A vector comprising a DNA sequence of claim 1 in operativeassociation with an expression control sequence for said DNA sequence.3. A host cell transformed with the vector of claim 2 said host cellsselected from the group consisting of mammalian, bacterial, yeast andinsect cells.
 4. A method for producing an osteoinductive protein, saidmethod comprising the steps of(a) culturing in a suitable culture mediumsaid transformed host cell of claim 3; and (b) isolating and purifyingsaid osteoinductive protein from said culture medium.
 5. The host cellof claim 4 wherein said host cell is a mammalian cell.
 6. The host cellof claim 4 wherein said cells are selected from the group consisting ofbacterial, yeast and insect cells.
 7. A mammalian host cell transformedwith the vector of claim 2.